P-selectin, also known as granule membrane protein-140 (GMP-140), or PADGEM protein, is an integral membrane glycoprotein found in secretory granules of both platelets and endothelial cells. See E. I. B. Peerschke, Am. J. Clin. Pathol., 98, 455 (1992). After activation of these cells by agonists such as thrombin, it is rapidly redistributed to the cell surface during degranulation. P-selectin belongs to the selectin family of vascular cell surface receptors that share sequence similarity and overall domain organization. See G. I. Johnston et al., Cell, 56, 1033 (1989). The other known selectins are ELAM-1, a cytokine-inducible endothelial cell receptor for neutrophils, and a leukocyte surface structure which plays a role in directing the homing of lymphocytes to high endothelial venules of peripheral lymph nodes. It has recently been shown by J.-G. Geng et al., Blood, 74, 65a (1989), that human neutrophils bind in a Ca.sup.2+ -dependent manner to purified P-selectin immobilized on plastic. Furthermore, adhesion of neutrophils to endothelium stimulated with rapid activators such as histamine is mediated at least in part by P-selectin. P-selectin is also involved in binding of activated platelets to monocytes and neutrophils. See, S. A. Hamburger et al., Blood, 75, 550 (1990) and E. Larsen, Cell, 59, 305 (1989).
Because platelet activation accompanies a number of vascular disorders such as unstable angina, peripheral vascular disease, stroke, and procedures such as angioplasty and coronary thrombolysis, considerable effort has been exerted during the last two decades to develop more sensitive and specific methods to detect activated, circulating platelets. See, for example, C. W. Hamm et al., J. Am. Coll. Cardiol., 10, 998 (1987); D. J. Fitzgerald et al., Circulation, 77, 142 (1988) and A. H. Gershlick, Circulation, 81, 128 (1991). The most reliable markers of in vivo platelet activation have been substances released from platelets after activation, which can be measured in the plasma or urine: platelet factor 4 (PF4), .beta.-thromboglobulin (.beta.-TG), and metabolites of thromboxane A.sub.2. These markers have not achieved widespread clinical acceptance, however, because of technical limitations pertaining to sample collection, processing, and analysis.
Several changes in surface membrane glycoprotein expression can be detected during platelet activation with specific murine monoclonal antibodies. For example, as reported by S. J. Shattil, in Blood, 70, 307 (1987), and C. S. Abrams et al., Blood, 75, 128 (1990), changes in the conversion of the GPIIb-IIIa complex to a functional fibrinogen receptor can be detected. J. N. George et al., J. Clin. Invest., 78, 340 (1986) reported that platelet activation with accompanying alpha granule release can be ascertained by examining P-selectin expression. Thus, assays have been designed that combine the use of activation-specific monoclonal antibodies with flow cytometry. See, for example, R. E. Scharf et al., Arteriosclerosis and Thrombosis, 12, 1475 (1992). These assays can be performed on whole blood and can facilitate the detection of platelet subpopulations that are heterogeneous with respect to their activation status. However, flow-cytometry requires expensive instrumentation, complex data processing and is not practical either to process large numbers of samples economically or to derive results within the timeframe required to affect clinical outcomes in acute situations such as those mentioned above.
Therefore, a need exists for a sensitive, simple and rapid in vitro assay to detect the extent of platelet activation.